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. 2019 Oct 16;294(47):17818–17836. doi: 10.1074/jbc.RA119.009521

Figure 5.

Figure 5.

Enhanced BMP signaling through ACVR1 promoted osteoclastogenesis. A, for TRAP staining, BMMs from control (caAcvr1) and caAcvr1 mutant (caAcvr1;Ubi-CreTM) mice were seeded in 48-well plates (1.5 × 104 cells/well) in the presence of M-CSF (20 ng/ml) and RANKL (50 ng/ml) for 5 days, and Cre activity was induced by 4-OHT (100 ng/ml) at the same time. For the demineralization assay, BMMs were seeded onto OsteoAssay plates (1.0 × 104 cells/well) and incubated for 7 days. Scale bars: 500 μm. B, TRAP-positive cells containing three or more nuclei were counted as osteoclasts. n = 12. C, number of nuclei per cell was analyzed. n = 12. D, relative number of small osteoclasts (i.e. those with three to five nuclei) and large osteoclasts (i.e. those with six or more nuclei) were quantified. n = 12. E, demineralized pits generated by osteoclasts was visualized by von Kossa staining. The pit area was measured using ImageJ. n = 4. F, after staining nuclei with DAPI, the total number of nuclei in culture was quantified during RANKL treatment from day 1 to day 5. n = 6. G, after culturing for 5 days, total RNA was extracted from osteoclasts, and the expression levels of osteoclast marker genes were assessed by qRT-PCR. n = 6 (control) and 5 (mutant). H and I, activation of Smad and non-Smad signaling was assessed by Western blotting. BMMs were cultured in the presence of M-CSF (20 ng/ml), RANKL (50 ng/ml), and 4-OHT (100 ng/ml) for 2 days. Differentiated osteoclasts were stimulated with BMP-2 (100 ng/ml) (H) and BMP-7(100 ng/ml) (I) for 30 min, and protein lysates were harvested. The protein levels of phosphorylated forms of p38, ERK, and AKT were normalized to the respective total form, and pSMAD1/5/9 levels were normalized to tSMAD1 using ImageJ. Fold increases of protein levels are relative to unstimulated control cells. Representative images of protein bands are shown. n = 3. Values represent the mean ± S.D. Differences were assessed by Student's t test and one-way ANOVA, followed by a Tukey test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.